Novel cellulase tolerant to surfactants

ABSTRACT

Disclosed is a novel cellulase having an amino acid sequence in which the 162nd and/or 166th amino acid residues in the amino acid sequence of cellulase NCE5 are substituted. Further, a polynucleotide encoding the novel cellulase, an expression vector containing the polynucleotide, a host cell transformed with the expression vector, and a cellulase preparation and a washing composition containing the cellulase are disclosed. The cellulase of the present invention is resistant to surfactants, and maintains a high activity even under alkaline conditions.

TECHNICAL FIELD

The present invention relates to a novel cellulase having an amino acidsequence wherein a partial amino acid sequence is substituted in thesequence of the original cellulase consisting of the amino acid sequenceof SEQ ID NO: 1.

BACKGROUND ART

Cellulases are utilized, based on their properties, in variousindustrial fields, particularly the field of fabric processing. In thisfield, a cellulose-containing fabric is treated with cellulase to impartdesired properties to the fabric. For example, a treatment withcellulase is carried out to improve the touch and/or appearance ofcellulose-containing fabric, or for a “biowash” which imparts a“stonewash” appearance to a colored cellulose-containing fabric, therebyproviding the fabric with localized color variations. Further, in theprocess for manufacturing lyocell, cellulase is used for removing thefuzz generated in the process from the fabric surface. In thisconnection, lyocell is a regenerated cellulose fabric derived from woodpulp, and has recently attracted attention for properties (such as highstrength or water absorption) and as a production process that causesless environmental pollution.

Hitherto, it has been considered that cellulase decomposes cellulose bythe synergistic effect of plural enzymes. Because of an evolution ofprotein separation techniques or genetic engineering techniques, anattempt has been carried out to separate enzyme components appropriatefor fabric processing from cellulase consisting of plural enzymes, andto produce the enzyme components. Particularly, cellulases derived frommicroorganisms belonging to filamentous fungi such as genus Trichodermaor genus Humicola have been subjected to serious study. For example, ascellulase components, CBH I, EG V, NCE4, and NCE2 in genus Humicola, andCBH I, CBH II, EG II, and EG III in genus Trichoderma were isolated, andthus, cellulase preparations containing as the major components one ormore specific cellulase components appropriate for each purpose can beproduced by preparing overexpressed enzymes or monocomponent enzymesusing genetic engineering techniques.

Further, it is known that endoglucanase enzyme NCE5 is useful inimparting a stonewash appearance to stained cellulose containing fiberor improving the touch thereof, as a cellulase which causes a slightredeposition or backstaining (i.e., a slight back staining) of an indigodye to clothing during the treatment (see patent reference 1).

In another aspect, when cellulases are used as a detergent for clothing,not only a quantitative improvement of cellulase components used butalso a qualitative one is desired. More particularly, a detergent forclothing contains various surfactants, and a solution obtained bysolubilizing the detergent for clothing in water is alkaline (pH10 topH11). Therefore, it is necessary that cellulases contained in adetergent for clothing should be resistant to various surfactants, andexhibit a strong activity under alkaline conditions.

-   (patent reference 1) International Publication WO01/90375 (pages    2-3, and the sequence of SEQ ID NO: 1 in the Sequence Listing)

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a novel cellulaseresistant to surfactants and/or having a high activity under alkalineconditions.

The present inventors conducted intensive studies, and as a result,successfully obtained a novel cellulase resistant to surfactants and/orhaving a high activity under alkaline conditions, by substituting the162nd and/or 166th amino acid residues with different amino acidresidues in the amino acid sequence of a cellulase (NCE5) consisting ofthe amino acid sequence of SEQ ID NO: 1. The present invention relatesto:

-   [1] a cellulase having (1) an amino acid sequence in which a 162nd    amino acid residue and/or a 166th amino acid residue in the amino    acid sequence of SEQ ID NO: 1 are substituted, or (2) an amino acid    sequence in which one or plural amino acids are added to or deleted    from the N-terminus of the amino acid sequence (1);-   [2] the cellulase of [1] having the amino acid sequence of SEQ ID    NO: 3;-   [3] the cellulase of [1], wherein the 166th amino acid is    substituted with glutamic acid or aspartic acid;-   [4] the cellulase of [3] having the amino acid sequence of SEQ ID    NO: 4;-   [5] a cellulase having the amino acid sequence of SEQ ID NO: 5;-   [6] a polynucleotide encoding the cellulase of [1] to [5];-   [7] an expression vector comprising the polynucleotide of [6];-   [8] a host cell transformed with the expression vector of [7];-   [9] a process for producing the cellulase of [1] to [5], comprising    the steps of:    -   cultivating the host cell of [8], and    -   collecting the cellulase from the host cell and/or culture        obtained by the cultivation;-   [10] a cellulase preparation comprising the cellulase of [1] to [5];-   [11] a washing composition comprising the cellulase of [1] to [5] or    the cellulase preparation of [10];-   [12] a method of treating a cellulose-containing fabric, comprising    the step of bringing the cellulose-containing fabric into contact    with the cellulase of [1] to [5], the cellulase preparation of [10],    or the washing composition of [11];-   [13] a method of reducing fuzzing of a cellulose-containing fabric    or reducing a rate of the formation of fuzz, comprising the step of    bringing the cellulose-containing fabric into contact with the    cellulase of [1] to [5], the cellulase preparation of [10], or the    washing composition of [11];-   [14] a method of reducing weight to improve the touch and appearance    of a cellulose-containing fabric, comprising the step of bringing    the cellulose-containing fabric into contact with the cellulase of    [1] to [5], the cellulase preparation of [10], or the washing    composition of [11];-   [15] a method of color clarification of a colored    cellulose-containing fabric, comprising the step of bringing the    colored cellulose-containing fabric into contact with the cellulase    of [1] to [5], the cellulase preparation of [10], or the washing    composition of [11];-   [16] a method of providing a localized color variation to a colored    cellulose-containing fabric, comprising the step of bringing the    colored cellulose-containing fabric into contact with the cellulase    of [1] to [5], the cellulase preparation of [10], or the washing    composition of [11];-   [17] a method of reducing stiffness of a cellulose-containing fabric    or reducing a rate of the formation of stiffness, comprising the    step of bringing the cellulose-containing fabric into contact with    the cellulase of [1] to [5], the cellulase preparation of [10], or    the washing composition of [11];-   [18] the method of [12] to [17], wherein the treatment of the fabric    is carried out by soaking, washing, or rinsing the fabric;-   [19] a method of deinking waste paper, comprising the step of    treating the waste paper with the cellulase of [1] to [5] or the    cellulase preparation of [10] together with a deinking agent;-   [20] a method of improving a freeness of paper pulp, comprising the    step of treating the paper pulp with the cellulase of [1] to [5] or    the cellulase preparation of [10]; and-   [21] a method of improving a digestibility of animal feed,    comprising the step of treating a cellulose-containing fabric with    the cellulase of [1] to [5] or the cellulase preparation of [10].

The term “polynucleotide” as used herein includes DNA and RNA.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be explained in detail hereinafter.

Original Cellulase

The term “original cellulase” as used herein means a cellulaseconsisting of the amino acid sequence of SEQ ID NO: 1. Further, asecretory signal sequence is sometimes processed differently, dependingon a host used for producing the original cellulase. Therefore, ahomologous protein having an amino acid sequence in which one or pluralamino acids are added to or deleted from the N-terminus of the aminoacid sequence of SEQ ID NO: 1 is included in the original cellulase.

Novel Cellulase of the Present Invention

As the novel cellulase in the present invention, when the originalcellulase consists of the amino acid sequence of SEQ ID NO: 1, there maybe mentioned, for example, a cellulase having an amino acid sequence inwhich one or two amino acid residues selected from the group consistingof the 162nd amino acid and the 166th amino acid in the amino acidsequence of SEQ ID NO: 1 are substituted with different amino acidresidues. Further, a cellulase obtained from an isolatednaturally-occurring strain is included in the cellulase of the presentinvention, so long as the 162nd and/or 166th amino acids in the aminoacid sequence of SEQ ID NO: 1, or the corresponding amino acid(s) aresubstituted.

As the cellulase of the present invention, a cellulase having an aminoacid sequence in which the 162nd amino acid in the amino acid sequenceof SEQ ID NO: 1 is substituted with proline and the 166th amino acidtherein is substituted with glutamic acid or aspartic acid, ispreferable. Further, a cellulase having the amino acid sequence of SEQID NOS: 3 to 5 is more preferable. The cellulases exhibit advantageousfeatures, that is, resistance to a surfactant and/or maintaining a highactivity under alkaline conditions.

As the novel cellulase in the present invention, when the originalcellulase is a homologous protein of the cellulase having the amino acidsequence of SEQ ID NO: 1, there may be mentioned, for example, acellulase having an amino acid sequence in which one or two amino acidresidues selected from the group consisting of amino acids correspondingto the 162nd amino acid and the 166th amino acid in the amino acidsequence of SEQ ID NO: 1 are substituted with different amino acidresidues. In this connection, the amino acid residues to be substitutedin the homologous protein may be easily selected by comparing amino acidsequences using a known algorithm.

Generation of Novel Cellulase of the Present Invention

The novel cellulase of the present invention may be generated, forexample, by recombinant DNA techniques or polypeptide synthesistechniques, or obtained from an isolated naturally-occurring strain.

When the recombinant DNA techniques are used, the novel cellulase of thepresent invention may be prepared, for example, by the followingprocedures. A DNA encoding an original cellulase is obtained, and asite-directed mutagenesis is carried out in the obtained DNA tosubstitute the desired amino acid(s). A host cell is transformed with anexpression vector comprising the mutated DNA, and the transformant iscultivated to prepare the novel cellulase. The DNA encoding an originalcellulase may be completely synthesized on the basis of the amino acidsequence of SEQ ID NO: 1 in consideration of a host cell used, and maybe preferably a DNA consisting of the nucleotide sequence of SEQ ID NO:2, which may be obtained from a cDNA library of Humicola insolens inaccordance with conventional methods used in genetic engineering.

Some methods for introducing a mutation into a specific site of a gene,such as a gapped duplex method [Methods in Enzymolog 154, 350(1987)] ora Kunkel method [Methods in Enzymology, 154, 367(1987)], are known tothose skilled in the art. These methods may be used for carrying out asite-directed mutagenesis in the DNA encoding an original cellulase. Thenucleotide sequence of the mutated DNA may be confirmed by, for example,a Maxam-Gilbert chemical modification method [Methods in Enzymology, 65,499(1980)] or a dideoxinucleotide chain termination method [Gene, 19,269(1982)]. The amino acid sequence of the cellulase of the presentinvention may be confirmed on the basis of the obtained nucleotidesequence.

Production of Novel Cellulase of the Present Invention

The cellulase of the present invention may be produced in a host cell bytransforming the host cell with a DNA fragment encoding the cellulase inthe form of a DNA molecule (particularly an expression vector) so thatthe DNA fragment may be replicated and expressed in the host cell.

According to the present invention, an expression vector comprising aDNA fragment encoding the cellulase of the present invention, so thatthe DNA fragment may be replicated and the protein encoded by the DNAfragment may be expressed in a host microorganism, is provided. Theexpression vector of the present invention can be constructed on thebasis of a self-replicating vector (such as a plasmid), which exists asan extrachromosomal element and can replicate independently of thereplication of chromosomes. Alternatively, the expression vector of thepresent invention may be a vector which is integrated into the genome ofthe host microorganism and replicated together with chromosomes, whenthe host is transformed with the vector. The construction of the vectorof the present invention can be carried out by ordinary procedures ormethods commonly used in genetic engineering.

To express a protein having a desired activity by transforming a hostmicroorganism with the expression vector, it is preferable that theexpression vector contains, for example, a DNA sequence capable ofcontrolling the expression, or a genetic marker to select transformants,in addition to the DNA fragment of the present invention. The DNAsequence capable of controlling the expression includes, for example, apromoter, a terminator, or a DNA sequence encoding a signal peptide forsecretion. The promoter is not particularly limited, so long as it showsa transcriptional activity in a host microorganism. The promoter can beobtained as a DNA sequence which controls the expression of a geneencoding a protein the same as or different from that derived from thehost microorganism. The signal peptide is not particularly limited, solong as it contributes to the protein secretion in a host microorganism.The signal peptide can be obtained as a DNA sequence derived from a geneencoding a protein the same as or different from that derived from thehost microorganism. The genetic marker can be appropriately selected inaccordance with the method for selecting a transformant. As the geneticmarker, for example, a drug resistance gene or a gene complementing anauxotrophic mutation can be used.

According to the present invention, a microorganism transformed with theexpression vector is provided. A host-vector system which can be used inthe present invention is not particularly limited. For example, a systemutilizing E. coli, Actinomycetes, yeasts, or filamentous fungi, or asystem for the expression of a fusion protein using such a microorganismcan be used. Transformation of a microorganism with the expressionvector can be carried out in accordance with an ordinary method in theart.

The transformant of the present invention is cultivated in anappropriate medium, and the resulting culture is used to obtain theisolated protein of the present invention. According to the presentinvention, a process for producing the novel protein of the presentinvention can be provided. The transformant can be cultivated under theconditions commonly used in the cultivation thereof. Further, after thecultivation, the protein of interest can be collected in accordance withan ordinary method in the art.

Use of Novel Cellulase of the Present Invention/Cellulase Preparation ofthe Present Invention

The present invention relates to a cellulase preparation comprising thecellulase of the present invention.

Conventionally, the cellulase preparation may contain, for example,fillers (for example, lactose, sodium chloride, or sorbitol),antiseptics, and/or nonionic surfactants, in addition to the cellulaseenzyme. The form of the cellulase preparation may be solid or liquid,such as powder, particulate, granule, non-dusting granule, or liquidformulation. In addition to the cellulase of the present invention, thecellulase preparation of the present invention may contain othercellulase enzymes, such as cellobiohydrolase, β-gulucosidase, orendoglucanase other than the endoglucanase of the present invention.

The non-dusting granule (preferably a granule not having a dustability),that is one form of cellulase preparation, can be produced according tothe common dry granulation method. That is, powder cellulase is mixedwith one or plural substances selected from the group comprisinginorganic salts such as sodium sulfate or sodium chloride which areneutral and do not have an effect on the endoglucanase activity;minerals such as bentonite or montmorillonite which do not have aneffect on the endoglunanase activity; and neutral organic substancessuch as starch or powder cellulase. Thereafter, the powders or thefinely suspended suspension of one or plural nonionic surfactants areadded to the mixture, and then the obtained product is fully mixed orkneaded. Depending on the situation, a synthetic polymer such aspolyethylene glycol or a natural polymer such as starch, which bindssolids, is optionally added to the mixture and further kneaded.Thereafter, granulation is carried out by extrusion molding, using, forexample, a disk pelleter, and the obtained molded material is thenconverted into a spherical form using a marumerizer followed by drying,so that non-dusting granules can be produced. The amount of one orplural nonionic surfactants is not particularly limited, and ispreferably 0.1 to 50% by weight, more preferably 0.1 to 30% by weight,most preferably 0.1 to 10% by weight to the total weight of thecellulase preparation of the present invention. It is also possible tocoat the surface of granules with a polymer or the like to control thepermeation of oxygen or water.

Further, the liquid preparation, which is one of the cellulasepreparations (preferably stabilized liquid), can be prepared by blendingan endoglucanase stabilizer (such as a synthetic or natural polymer)with a solution containing the cellulase of the present invention and,if necessary, adding inorganic salts and/or a synthetic preservative. Inthis case, one or plural nonionic surfactants can be blended with theliquid preparation. The amount of one or plural of the nonionicsurfactants is not particularly limited, and is preferably 0.1 to 50% byweight, more preferably 0.1 to 30% by weight, most preferably 0.1 to 10%by weight to the total amount of the cellulase preparation of thepresent invention.

Further, the present invention provides a washing composition comprisingthe cellulase of the present invention or the cellulase preparation ofthe present invention. The washing composition may also comprisesurfactants which may be anionic, nonionic, cationic, amphoteric orzwitterionic, or a mixture thereof. The washing composition may compriseother washing compositions known in the art, for example, a builder,bleach, bleaching agent, tarnish inhibitor, sequestant, soil releasingpolymer, flavor, other enzymes (such as protease, lipase, or amylase),stabilizer for enzyme, granulater, optical brightner, and/or foamingagent. As typical anionic surfactants, there may be mentioned, forexample, linear alkyl benzene sulfonate (LAS), alkyl sulphate (AS),α-olefin sulfonate (AOS), polyoxyethylene alkylether sulfonate (AES),α-sulfo fatty acid ester (α-SFMe), or alkali metal salts ofnaturally-occurring fatty acid. As the nonion surfactants, there may bementioned, for example, polyoxyethylene alkyl ether (AE),alkylpolyethylene glycol ether, nonylphenol polyethylene glycol ether,fatty acid methyl ester ethoxylate, sucrose, or fatty acid ester ofglucose, or esters of alkylglucoside or polyethoxylated alkylglucoside.

The method of the present invention for treating a cellulose-containingfabric is carried out by bringing the cellulose-containing fabric intocontact with the cellulase of the present invention, the cellulasepreparation of the present invention, or the washing composition of theinvention.

The following properties of cellulose-containing fabric can be improvedby the method of the present invention:

-   (1) Removal of fuzz (reduction of the rate of the formation of fuzz,    and reduction of fuzz);-   (2) Improvement of the touch and appearance of a fabric by reducing    weight;-   (3) Color clarification of a colored cellulose-containing fabric;-   (4) Providing a localized color variation to a colored    cellulose-containing fabric, that is, providing a stonewash-like    appearance and texture to a colored cellulose-containing fabric,    typically jeans; and-   (5) Softening of a fabric (reduction of the rate of stiffness, and a    reduction of stiffness).

More particularly, the method of the present invention can be carriedout by adding the cellulase of the present invention, the cellulasepreparation of the present invention, or the washing composition of thepresent invention into water in which a fabric is or will be soaked, forexample, during a soaking, washing, or rinsing of a fabric.

Conditions such as contact temperature or the amount of the cellulose,the cellulase preparation, or the washing composition may beappropriately determined in accordance with various other conditions.For example, when reducing the rate of the formation of fuzz or reducingfuzzing of the cellulose-containing fabric, the cellulase, the cellulasepreparation, or the washing composition in a protein concentration of0.01 to 20 mg/L is preferably used at a temperature of approximately 10to 60° C.

In a processing of reducing weight to improve the touch and appearanceof the cellulose-containing fabric, the cellulase, the cellulasepreparation, or the washing composition in a protein concentration of0.1 to 50 mg/L is preferably used at a temperature of approximately 10to 60° C.

When clarifying the color of the colored cellulose-containing fabric,the cellulase, the cellulase preparation, or the washing composition ina protein concentration of 0.01 to 20 mg/L is preferably used at atemperature of approximately 10 to 60° C.

When providing a localized color variation to a coloredcellulose-containing fabric, the cellulase, the cellulase preparation,or the washing composition in a protein concentration of 0.1 to 100 mg/Lis preferably used at a temperature of approximately 20 to 60° C.

When reducing the stiffness of a cellulose-containing fabric or reducingthe rate of the formation of stiffness, the cellulase, the cellulasepreparation, or the washing composition in a protein concentration of0.01 to 20 mg/L is preferably used at a temperature of 10 to 60° C.

Further, the present invention relates to a method for deinking wastepaper, characterized by using the cellulase of the present invention orthe cellulase preparation of the present invention, in the process oftreating the waste paper together with a deinking agent.

The cellulase or the cellulase preparation of the present invention isuseful in the process of producing recycled paper from waste paper,since an efficiency of the deinking can be improved by reacting wastepaper therewith. According to the deinking method, the whiteness ofwaste paper can be improved by remarkably reducing residual-ink fabric.

The deinking agent is not particularly limited, so long as it is agentwhich can be used in deinking waste paper in general. As the deinkingagent, there may be mentioned, for example, alkalis (such as sodiumhydroxide or sodium carbonate), sodium silicate, hydrogen peroxide,phosphates, anionic or nonionic surfactants, scavengers such as oleicacid, and assistant agents such as a pH stabilizer, a chelating agent,or a dispersing agent.

Waste paper which can be treated by the deinking method is notparticularly limited, so long as it is common waste paper. As the wastepaper, there may be mentioned, used newspaper, used magazine paper, andlow to middle grade printed used paper which comprises mechanical pulpand chemical pulp; used wood-free paper comprising chemical pulp; orprinted waste paper thereof such as coating paper. A paper other thanthe common waste paper can be treated by the deinking method, so long asit deposits ink.

Further, the present invention relates to a method for improving afreeness of paper pulp, which comprises the process of treating a paperpulp with the cellulase of the present invention or the cellulasepreparation of the present invention.

According to the method, it is considered that this method cansignificantly improve a freeness of paper pulp, without a decline ofstrength. A paper pulp which can be treated by the method is notparticularly limited, but there may be mentioned, for example, wastepaper pulp, recycled paperboard pulp, kraft pulp, sulfite pulp,thermo-mechanical treatment pulp, and other high-yield pulp.

Further, the present invention relates to a method for improving adigestibility of animal feed, comprising the step of treating the animalfeed with the cellulase of the present invention or the cellulasepreparation of the present invention. According to this method, adigestibility of animal feed can be improved by digesting glucan inanimal feed into appropriate molecules having a low molecular weight.

Further, a digestibility of glucan in animal feed can be improved byusing the cellulase of the present invention in animal feed. Accordingto the present invention, a method for improving a digestibility ofanimal feed, comprises the step of treating the animal feed with thecellulase of the present invention or the cellulase preparation of thepresent invention.

As shown above, the cellulase and the washing composition of the presentinvention and the method of the present invention using the same havebeen described. International Publication WO01/90375 discloses that anoriginal cellulase of the present invention, cellulase NCE5, can be usedas an active ingredient of the cellulase preparation or the washingcomposition; that cellulase NCE5 or a cellulase preparation or washingcomposition containing cellulase NCE5 can improve various properties ofa cellulose-containing fabric [for example, removal of fuzz (such as, areduction of the rate of the formation of fuzz, and reduction of fuzz);improvement of the touch and appearance of a fabric by reducing weight;color clarification of a colored cellulose-containing fabric; providinga localized color variation to a colored cellulose-containing fabric; orsoftening of a fabric (such as, reduction of the rate of the stiffness,and reduction of stiffness)]; that cellulase NCE5 or a cellulasepreparation or washing composition containing cellulase NCE5 can deinkwaste paper; that cellulase NCE5 or a cellulase preparation or washingcomposition containing cellulase NCE5 can improve a freeness of paperpulp; and that cellulase NCE5 or a cellulase preparation or washingcomposition containing cellulase NCE5 can improve a digestibility ofanimal feed. The cellulase of the present invention exhibits anexcellent resistance to surfactants and alkaline conditions, comparedwith the original cellulase NCE5. Therefore, it can be expected thatadvantageous effects may be obtained when the cellulase is used in theabove methods using cellulase NCE5, or used as an active ingredient ofthe cellulase preparation or washing composition, instead of NCE5.

Deposition of Microorganism

Escherichia coli JM109 transformed with plasmid pNCE5Bam comprising aDNA which encodes an original cellulase of the present invention wasdomestically deposited (FERM BP-7138) in the International PatentOrganism Depositary National Institute of Advanced Industrial Scienceand Technology [(Former Name) National Institute of Bioscience andHuman-Technology Agency of Industrial Science and Technology (Address:AIST Tsukuba Central 6, 1-1, Higashi 1-chome Tukuba-shi, Ibaraki-ken305-8566 Japan)] on Apr. 18, 2000.

Humicola insolens FERM BP-5977, which was used in Example 2(2), wasdomestically deposited (FERM P-15736) in the International PatentOrganism Depositary National Institute of Advanced Industrial Scienceand Technology on Jul. 15, 1996, and was transferred to an internationaldeposit (FERM BP-5977) on Jun. 13, 1997.

EXAMPLES

The present invention now will be further illustrated by, but is by nomeans limited to, the following Examples.

All changes and modifications not mentioned in the present specificationbut obvious to those skilled in the art are herein incorporated byreference.

Example 1 Construction of Genes Encoding Cellulases

DNAs encoding a cellulase [hereafter referred to as A162P (SEQ ID NO:3)] in which the 162nd amino acid of the original cellulase consistingof the amino acid sequence of SEQ ID NO: 1 was substituted with proline,a cellulase [hereafter referred to as K166E (SEQ ID NO: 4)] in which the166th amino acid thereof was substituted with glutamic acid, and acellulase [hereafter referred to as APKE (SEQ ID NO: 5)] in which the162nd and 166th amino acids thereof were substituted with proline andglutamic acid, respectively, were constructed in accordance with thefollowing procedures.

The desired site-directed mutations were introduced into a DNA encodingthe original cellulase by using plasmid pNCE5Bam (WO 01/90375) as atemplate DNA, and LA PCR in vitro Mutagenesis Kit (Takara Shuzo), inaccordance with a protocol attached to the kit. Three primers were usedfor the site-directed mutagenesis. (SEQ NO: 6) NCE5-A162P:5′-GGGGAAGGGGTCGCACTCGTGGCGTTG-3′ (SEQ NO: 7) NCE5-K166E:5′-CTTGAGCTCCTCGGGGAAGGCGTCGCA-3′ (SEQ NO: 8) NCE5-APKE:5′-GAGCTCCTCGGGGAAGGGGTCGCACTCGTG-3′The primers NCE5-A162P, NCE5-K166E, and NCE5-APKE are primers forgenerating the DNAs which encode the cellulases A162P, K166E, and APKE,respectively.

The mutation-introduced PCR products obtained by using the three primerswere digested with restriction enzymes EcoRI and PstI, and extractedwith phenol:chloroform:isoamyl alcohol (25:24:1), and then ethanolprecipitation was carried out. Each DNA fragment was ligated to plasmidpUC118 previously digested with restriction enzymes EcoRI and PstI, toobtain plasmids pNCE5AP-118(A162P), pNCE5KE-118 (K166E), andpNCE5APKE-118 (APKE). The nucleotide sequence of each fragment insertedin the three plasmids was determined by a fluorescent DNA sequencer (ABIPRISM 310 Genetic Analyzer; Perkin-Elmer) to confirm that only thedesired mutations were introduced into the DNA fragments.

Example 2 Expression of each Cellulase Gene in Humicola insolens

(1) Construction of Plasmids for Expressing Cellulase Genes

The plasmids pNCE5AP-118, pNCE5KE-118, and pNCE5APKE-118 comprising eachcellulase gene obtained in Example 1 were digested with restrictionenzyme BamHI, and were subjected to agarose gel electrophoresis tocollect three DNA fragments of approximately 0.7 kbp. Each DNA fragmentwas ligated to expression vector pJD-c5 (WO 01/90375) for Humicolainsolens FERM BP-5977, which was previously digested with restrictionenzyme BamHI and dephosphorylated with alkaline phosphatase (TakaraShuzo) derived from E. coli, to obtain plasmids pNCE5AP(A162P),pNCE5KE(K166E), and pNCE5APKE(APKE) for expressing each cellulase gene.

(2) Transformation of Humicola insolens FERM BP-5977

Humicola insolens FERM BP-5977 was cultivated in an (S) medium at 37° C.for 24 hours, and collected by centrifugation at 3000 rpm for 10 min.The (S) medium was prepared by removing avicel from a (N) medium (5.0%avicel, 2.0% yeast extract, 0.1% peptone, 0.03% calcium chloride, 0.03%magnesium chloride, pH 6.8) and adding glucose (3.0%) thereto. Theresulting mycelia were washed with 0.5 mol/L sucrose, and suspended in10 mL of an enzyme solution for generating protoplasts (3 mg/mLglucuronidase, 1 mg/mL Chitinase, 1 mg/mL Zymolyase, and 0.5 mol/Lsucrose) previously filtrated with a filter (0.45 μm). The suspensionwas shaken at 30° C. for 60 to 90 minutes to generate protoplasts. Thesuspension was filtrated, and centrifuged at 2500 rpm for 10 minutes tocollect protoplasts. The protoplasts were washed with an SUTC buffer[0.5 mol/L sucrose, 10 mmol/L calcium chloride, and 10 mmol/L Tris-HCl(pH 7.5)].

The protoplasts were suspended in 1 mL of the SUTC buffer. To 100 μL ofthe suspension, 10 μg of each DNA (TE) solution (10 μL) was added, andallowed to stand on ice for 5 minutes. Further, 400 μL of a PEG solution[60% PEG4000, 10 mmol/L calcium chloride, and 10 mmol/L Tris-HCl (pH7.5)] was added, and allowed to stand on ice for 20 minutes. After 10 mLof the SUTC buffer was added, the whole was centrifuged at 2500 rpm for10 minutes. The collected protoplasts were suspended in 1 mL of the SUTCbuffer, centrifuged at 4000 rpm for 5 minutes, and finally suspended in100 μL of the SUTC buffer.

The protoplasts treated as above were overlaid with YMG [1% glucose,0.4% yeast extract, 0.2% malt extract, and 1% agar (pH 6.8)] soft agaron a YMG medium containing hygromycin (200 μg/mL), and incubated at 37°C. for 5 days to obtain transformants as colonies.

(3) Evaluation of Transformants by SDS-PAGE

Humicola insolens transformants obtained by using plasmids pNCE5AP,pNCE5KE, and pNCE5APKE were cultivated in the (N) medium (5.0% avicel,2.0% yeast extract, 0.1% peptone, 0.03% calcium chloride, 0.03%magnesium chloride, pH 6.8) at 37° C. for 5 days, and the resultingculture supernatants were analyzed by SDS-PAGE.

The SDS-PAGE was carried out by using a Tefco system composed of a tankfor electrophoresis (No. 03-101), a power supply (Model: 3540), 12% gel(01-005), and a buffer kit for SDS-PAGE (06-0301), at 18 mA/90 min.After the electrophoresis, the gel was stained with a Coomassiebrilliant blue R-250 solution (0.1% Coomassie brilliant blue R-250, 40%methanol, and 10% acetic acid), and decolorized with a decoloringsolution (10% methanol and 7.5% acetic acid) to detect proteins. An LMWMarker Kit (17-0446-01; Amersham Bioscience) was used as a molecularweight marker.

As a result, transformants in which an expression of a 25 kDa proteinwas enhanced were found, and it was confirmed that the desired cellulasewas produced in the transformants. Among the transformants, strainsK215-40, K215-42, and K229-72 were selected as the transformantsproducing the cellulase A162P, K166E, and APKE, respectively, and wereused in further analysis. Further, the original cellulase NCE5 describedin WO01/90375 was used as a control.

Example 3 Evaluation of Cellulases Obtained by Each Transformant

Culture supernatants obtained by cultivating Humicola insolenstransformants K215-40, K215-42, and K229-72 in the (N) medium at 37° C.for 5 days were used to measure an EGU activity in three experimentalgroups, that is, (1) pH 6.0, (2) pH 6.0 and addition of 200 ppm sodiumlinear alkyl benzene sulfonate (LAS), and (3) pH 10.0.

The EGU activity was measured in accordance with the followingprocedures. Carboxymethyl cellulose (Hercules) was dissolved in anappropriate buffer to a final concentration of 3.5% as a substratesolution. That is, a substrate solution for the measurement at pH 6.0was prepared by dissolving the substrate in a 0.1 mol/L phosphate buffer(pH 6.0), and a substrate solution for the measurement at pH 10.0 wasprepared by dissolving the substrate in a 0.1 mol/L Tris-HCl buffer (pH10.0). After 5 mL of each substrate solution was added to a test tube,each test tube was preheated in a water bath at 40° C. for 10 minutes.To the test tube, 0.15 mL of each test solution was added and the wholewas mixed well to start an enzyme reaction at 40° C. After the reactionwas carried out for 30 minutes, a viscosity of each reaction solutionwas measured by an R-type viscometer (RE 100; Toki Sangyo) in which thetemperature was set at 40° C. An amount of enzyme which reduces theinitial viscosity to ½ under each enzyme reaction condition is definedas “1 unit (U)”. In Table 1, enzyme activities in the experimentalgroups (2) and (3) are shown as relative values when enzyme activitiesin the experimental group (1) are regarded as 100%. TABLE 1 Experimentalgroup Test sample (1) (2) (3) K215-30 (A162P) 100 26.6 29.6 K215-42(K166E) 100 26.7 29.5 K229-72 (APKE) 100 21.1 36.6 NCE (original 100 7.621.5 cellulase)

As shown in Table 1, it was found that each cellulase (A162P, K166E, andAPKE) was resistant to LAS and maintained a high activity under alkalineconditions, in comparison with the original cellulase.

As a result, on comparison of the original cellulase, it was found thatthe each cellulase (A162P, K166E, and APKE) had a resistance to the LASand a high activity under alkaline conditions.

INDUSTRIAL APPLICABILITY

The novel cellulase of the present invention is resistant to surfactantsand exhibits a higher activity under alkaline conditions, in comparisonwith the original cellulose, and thus is useful as an active ingredientof a detergent for clothing.

Free Text in Sequence Listing

Each amino acid sequence of SEQ ID NOS: 3 to 5 is that of a cellulaseresistant to surfactants. Each nucleotide sequence of SEQ ID NOS: 6 and7 is a primer for site-directed mutagenesis.

Although the present invention has been described with reference tospecific embodiments, various changes and modifications obvious to thoseskilled in the art are possible without departing from the scope of theappended claims.

1. A cellulase having (1) an amino acid sequence in which a 162nd aminoacid and/or a 166th amino acid in the amino acid sequence of SEQ ID NO:1 are substituted, or (2) an amino acid sequence in which one or pluralamino acids are added to or deleted from the N-terminus of the aminoacid sequence (1).
 2. The cellulase according to claim 1 having theamino acid sequence of SEQ ID NO:
 3. 3. The cellulase according to claim1, wherein the amino acid at the 166th position is substituted withglutamic acid or aspartic acid.
 4. The cellulase according to claim 3having the amino acid sequence of SEQ ID NO:
 4. 5. A cellulase havingthe amino acid sequence of SEQ ID NO:
 5. 6. A polynucleotide encodingthe cellulase according to claim
 1. 7. An expression vector comprisingthe polynucleotide according to claim
 6. 8. A host cell transformed withthe expression vector according to claim
 7. 9. A process for producingthe cellulase according to claim 1, comprising the steps of: cultivatingthe host cell and collecting the cellulase from the host cell and/orculture obtained by the cultivation.
 10. A cellulase preparationcomprising the cellulase according to claim
 1. 11. A washing compositioncomprising the cellulase according to claim 1 or the cellulasepreparation.
 12. A method of treating a cellulose-containing fabric,comprising the step of bringing the cellulose-containing fabric intocontact with the cellulase according to claim 1, the cellulasepreparation, or the washing composition.
 13. A method of reducingfuzzing of a cellulose-containing fabric or reducing a rate of theformation of fuzz, comprising the step of bringing thecellulose-containing fabric into contact with the cellulase according toclaim 1, the cellulase preparation, or the washing composition.
 14. Amethod of reducing weight to improve the touch and appearance of acellulose-containing fabric, comprising the step of bringing thecellulose-containing fabric into contact with the cellulase according toclaim 1, the cellulase preparation, or the washing composition.
 15. Amethod of color clarification of a colored cellulose-containing fabric,comprising the step of bringing the colored cellulose-containing fabricinto contact with the cellulase according to claim 1, the cellulasepreparation, or the washing composition.
 16. A method of providing alocalized color variation to colored cellulose-containing fabric,comprising the step of bringing the colored cellulose-containing fabricinto contact with the cellulase according to claim 1, the cellulasepreparation, or the washing composition.
 17. A method of reducingstiffness of a cellulose-containing fabric or reducing a rate of theformation of stiffness, comprising the step of bringing thecellulose-containing fabric into contact with the cellulase according toclaim 1, the cellulase preparation, or the washing composition.
 18. Themethod according to claim 12, wherein the treatment of the fabric iscarried out by soaking, washing, or rinsing the fabric.
 19. A method ofdeinking waste paper, comprising the step of treating the waste paperwith the cellulase according to claim 1 or the cellulase preparationtogether with a deinking agent.
 20. A method of improving a freeness ofpaper pulp, comprising the step of treating the paper pulp with thecellulase according to claim 1 or the cellulase preparation.
 21. Amethod of improving a digestibility of animal feed, comprising the stepof treating the animal feed with the cellulase according to claim 1 orthe cellulase preparation according to.